Composition for dietary enrichment

ABSTRACT

The present invention relates to a composition comprising one or more carotenoid, an aqueous diluent, and one or more of the following substances: vitamins, minerals, amino acids, fats or polysaccharides, wherein the composition is in the liquid state. In particular, the composition is for enriching animals&#39; dietary intake. The invention also includes enriched feed and methods for enriching feed.

The present invention relates to compositions for enriching animals'dietary intake. Compositions of this type are particularly useful forenriching animal feed, in particular, feed for fish, crustacea andpoultry. The invention includes enriched feed and methods for enrichingfeed.

Animals that are kept as pets, in zoos or that are used in the farmingindustry are kept captive, away from their natural habitat. One of theinherent problems with keeping animals in such an environment is theneed to provide them with a diet that adequately reflects thenutritional diversity and bioavailability of their natural diet. Failureto provide the proper dietary requirements results in negative effectson growth, reproduction, health and sustainability of a captivepopulation.

The basic nutrition for captive species is normally provided by live ordead (whole or part) animals, plant matter, or a variety of processedfeeds that may come in a variety of forms, such as pellets, flakes,biscuits etc.

However, a diet based purely on such food is often not sufficient toprovide an animal with its total dietary requirement. Additionally,harvesting, processing, manufacture and storage of food can lead to areduction in the nutritional value of the food. Exposure to light, heat,pressure, mechanical actions, atmospheric conditions or irradiation alsodamages feed ingredients resulting in reduced quantities of nutrientsand/or reduced bioavailability of important dietary components.Nutrients that may be affected include fats, vitamins, and carotenoids.For example, the most commonly used frozen marine feeds (TMC Brineshrimpand Mysis) generally have poor pigment profiles due to processing and assuch need supplementing with an external carotenoid source.

Every species requires a full complement of their essential vitamins,minerals, fatty acids and amino acids in their diet, in addition toenergy which can be derived from polysaccharides or lipids. Maintaininga proper dietary balance of, for example, fat and protein is essentialfor health of animals.

Minerals are required in the diet of many species for use in a number ofbiological processes involving metalloenzymes, neurotransmitters, oxygencarrying compounds, and skeletal structure.

Lipids are required not only as an energy source but also are essentialfor the synthesis of phospholipids, steroids and structural elements incell walls.

Carotenoids are also considered to be an important dietary component formany species. Carotenoids are pigments that are known to act as powerfulantioxidants. Certain carotenoids are additionally known to providepigmentation and coloration of animal tissues. For example, a redcarotenoid pigment can be added to the diet of broiler chickens tocolour the shanks, and to the diet of farmed trout to produce the samebrightly coloured flesh as seen in wild trout.

Peptides and nucleotides have been shown to increase nutrient and drugabsorption and lead to beneficial effects in growth rates and health.Peptides and nucleotides are also known to alter the absorptive area ofthe intestinal mucosa in fish.

Accordingly, it is common to supplement basic feeds with a number ofadditional substances.

However, conventional supplements do not properly counter deficienciesin the basic feed of the animals, often not providing the proper rangeand composition of components required for a balanced diet. Componentsof the supplements have also been shown to have a low level ofbioavailability and so are of little worth in enriching the diet of ananimal.

Thus, it is an object of the present invention to develop a compositionfor use in enriching an animal's diet that does not possess theaforementioned disadvantages of previously identified compositions.

It has been surprisingly discovered that the composition of the presentinvention provides an enhanced level of enrichment of an animals diet,as well as providing a high level of bioavailability. The general healthof an animal ingesting feed enriched by the inventive composition hasbeen shown to improve, including an improvement in healing and reductionin pathogen loading. For example, veterinary records for fish receivingfeed enriched with the composition show a reduction in the prevalence ofpathology and diseases affecting the skin. Also noted in clinicalassessments of such fish is a noticeable reduction in pathogen loadingwithin the mucous coat of the skin and fins and an increase in tissuehealing rates.

Additionally, the composition affords stability to the active componentsduring storage, application and the post application period. Thecomposition can be stored for over 18 months at typical storagetemperatures for such products ensuring target enrichment of feed at allstages of the product life. Additionally, this composition is stablewhen incorporated into feed for longer than current commerciallyavailable feed enrichment products such as Carophyll Pink CWD.

Accordingly, in a first aspect of the present invention, there isprovided a composition comprising one or more carotenoids, and one ormore of the following substances: vitamins, minerals, amino acids,lipids, peptides, nucleotides and/or polysaccharides.

A particular surprising advantage of such compositions is the ability,when incorporated in an animal's diet, to maintain and/or restorenatural skin colour and health in the animal. It is conventional forfeed supplements to be added to feed in order to alter the colour of theflesh. However, maintaining or restoring colour to the skin of theanimal by supplementing the animals feed has always proven to bedifficult in the past.

It is know that fish provided with traditional synthetic carotenoidsources do not develop or maintain full natural skin coloration, lustreand health. As an example, clown fish fed on a granulated dietcontaining 1000 mg/kg synthetic astaxanthin do not to significantlyalter skin coloration as compared to fish fed on their standard basediet. However, it has been shown that when the clown fish diet issupplemented with the compositions of the present invention the skincolour and definition of skin colour regions is dramatically improved.Amazingly, this improvement was noted at carotenoid levels of below 50mg/kg (astaxanthin weight/final feed weight).

In a number of cases, skin colour and health have been noted byveterinary and visual examination to have significantly improved withintwo weeks of commencement of feed supplementation. Natural colourenhancement has been noted without specific colours being limited. Goodlong term fish health, colour maintenance and restoration of deficientcolour have been noted at food enrichment levels of between 4 and 12mg/kg astaxanthin in enriched feed.

A similar enhancement of colouration has been found in invertebrates andreptiles.

This composition may be prepared for administration in a number of ways.

For example, the composition may be given directly in the liquid form,as an encapsulated liquid preparation, or incorporated in the feed inliquid form.

Thus, in a further preferred embodiment, the composition comprises anaqueous diluent and is preferably in the liquid state.

It should be understood that any aqueous diluent may be used that couldbe ingested, without experiencing toxic effects, by the species that isintended to consume the composition. Preferably, the aqueous diluent iswater, most preferably the aqueous diluent is purified water.

It has been found that the liquid form of composition is particularlyeffective, especially when given as an encapsulated liquid or addeddirectly to enrich feeds.

Encapsulation techniques are known in the art and may comprise a centralreservoir of the composition surrounded by a protective capsule, thematrix of the capsule preferably contains antioxidants.

The direct enrichment of feed is achieved by adding the composition tofeeds during or post manufacture, harvesting, processing, or delivery tothe consumer.

Conventionally, dry powdered vitamin, mineral, carotenoid and amino acidetc. preparations are used to enrich feeds. However, the use of suchpreparations has a number of distinct disadvantages.

It is virtually impossible to produce uniformly enriched foods usingsuch powdered particles, or fine aggregates. These preparations have alow level of adherence to the feed. Since the powdered particles tend tobe small in comparison to the feed, the preparations are susceptible topost enrichment settlement, thereby producing a variance in feedquality, especially following storage, transport and distribution.

These enrichment compositions also suffer from the same problems as thebasic feed, in that exposure to light, heat, pressure, mechanicalactions, atmospheric conditions or irradiation can damage compositions,thereby reducing the value of the enrichment.

It has been found that liquid compositions simplify the enrichmentprocess, provide an enhanced uniform distribution and adherence to thefeed, as well as providing a high level of bioavailability.Additionally, the composition affords stability to the active componentsduring storage, application and the post application period.

In a preferred embodiment one or more of the components of thecomposition are water soluble.

In a further preferred embodiment one or more of the components of thecomposition are fat soluble. Preferably, the fat soluble components areprovided in micelles.

It has been found that the ability to enrich feed with the compositionof the present invention can be enhanced by providing the composition inthe form of an emulsion or dispersion. In particular it has been shownthat providing one or more of the fat soluble components (particularlycarotenoids) of the composition in the form of a micelle allows aconvenient and highly efficient preparation for administering thecomposition. Not wishing to be bound by theory, it would appear that themicelle structure offers a high level of stability for the lipid solublecomponents and high level of absorption and retention in the feedbecause of the micelle structure having a high affinity for fats in thefeed, thereby ensuring the composition is not lost from the feed. Thisis particularly important when the enriched feed is delivered to thetarget animal in an aquatic environment.

The absorption and retention of such compositions is particularlyevident in crustaceans where a high level of unsaturated fats includingwaxy esters are present. A good level of absorption and retention of thecomposition by feed such as live juvenile crustacea is particularlyimportant since such feed do not have developed mouth parts and so cannot depend on ingestion to load the composition with the body.

Accordingly, in a preferred embodiment of the current invention, whenthe composition comprises an aqueous diluent and is in the liquid statethe fat soluble components are in the form of micelles.

However, the liquid form is not the only form the composition may take.

In a further preferred embodiment of the invention, the composition isformed into a tablet, or microencapsulated preparation, preferably thesecompositions do not contain a liquid diluent. Microencapsulatedpreparations are known in the art and usually comprise a core of thecomposition covered by a protective matrix, preferably the matrixincludes antioxidants. The tablet or microencapsulated preparation mayeither be ingested in isolation from the feed or ingested along withfeed. Often it is desirable to hide the tablets or microencapsulatedpreparations or tablets in the feed so that the animal unknowinglyingests the tablet. The tablet or microencapsulated product may also beprepared for dissolving in a liquid diluent prior to ingestion.

The choice of carotenoid, vitamin, mineral, amino acid, lipid, peptide,nucleotide or polysaccharide is dictated by the particular species andage of the animal intended to ingest the composition, and thedeficiencies in their diet. Accordingly, the skilled person would beable to determine the appropriate carotenoid, vitamin, mineral, aminoacid, lipid, peptide, nucleotide or polysaccharide in these specificcircumstances.

Not wishing to be limited further, but in the interests of clarity, thefollowing are examples of suitable components of the compounds of theinvention.

Examples of suitable carotenoids are those derived from yeast (e.g.Phaffia rhodozyma) or algae (e.g. Haematoccocus algae), extracted fromoleoresins, lucantin pink or astaxanthin glucosides. Preferably thecarotenoid is astaxanthin esterified to fatty acid acyl groups, suchcarotenoids show surprising absorption properties, particularly in feedscontaining high lipid levels (e.g. krill, mysis and brineshrimp).Preferably the water soluble carotenoid is an astaxanthin glucoside.When coloration of the target animal is required specific carotenoidsmay be chosen in order to enhance specific colours.

Examples of suitable vitamins are A, B1, B2, B6, B12, C (vitamin C maybe included as ascorbyl polyphosphate), D, E, K, Nicotinamide, Choline,Inositol, folic acid and Biotin. Preferably, the fat soluble vitaminsare A, D, E and K. Preferably, the water soluble vitamins are C, B1, B2,B6, B12, Nicotinamide, Choline, Inositol, folic acid or Biotin.

Examples of suitable minerals are iodide, iron, manganese, calcium,phosphorous, sodium, potassium, magnesium, zinc, copper or selenium.

Preferably, the amino acids are the essential amino acids for the animalthat is to ingest the composition. However, non-essential amino acidsare also contemplated for inclusion in the composition of the inventionsince it has been shown that their inclusion reduces the quantitativerequirement for essential amino acids. For example, the essential aminoacids for salmonid fish, and appropriate for including in thecomposition of the invention, are arginine, histidine, isoleucine,leucine, lysine, methionine phenylalanine, threonine, tryptophan andvaline. Non-essential amino acids cysteine and tyrosine ate alsosuitable amino acids. It has been shown in salmonid fish that cysteinecan replace up to a third of the required methionine and tyrosine canreplace up to a fifth of the required phenylalanine. Some amino acidshave also been shown to act as feeding behaviour modifiers. For example,in carnivorous fish the following compounds have been shown to alterfeeding responses: glycine, proline, taurine, valine, betanea andinosine. These amino acids are also contemplated as being suitable forinclusion in the claimed composition.

A variety of lipids and lipid derived compounds may be included in thecomposition. Preferably, the lipids are fats and more preferably oilswhich may be added along with one or more carotenoid as an oleoresin. Abalanced addition of oils of suitable chain length have been found toaid enrichment. However, the lipids may also be fatty acids,triglycerides, phospholipids and other neutral lipids such asalkyldiacylglycols, sterol esters, wax esters and pigments. Examplesinclude but are not restricted to: phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,plasmalogens, sphingomyelin, cerebrosides and gangliosides.

Thus, essential fatty acids may be added to the composition. Fish andterrestrial mammals do not possess the desaturase enzymes necessary tosynthesize 18:2ω6 or 18:3ω3 fatty acids and so these fatty acids must beadded to the diet to maintain cellular function and normal growth.

Waxy esters or their precursors may be added to the composition toincrease the availability of these important dietary components in somespecies. Waxy esters are esters of a fatty acid and a long-chain fattyalcohol. Crustaceans and some fish contain high levels of wax esterssuch as those comprising fatty acids esterified to hexadecanol.Therefore, the composition would preferably include wax esterscomprising fatty acids esterified to hexadecanol.

The lipids may be included from a variety of chain lengths, preferablyC14-C25. These may include but are not restricted to; C14, C16, C18,C20, C22, C25.

Examples of lipids which may be added to the composition include but arenot restricted to; 14:0, 16:0, 16:1, 18:0, 18:1ω9, 18:2ω6, 18:3ω3,18:4ω3, 20:1ω9, 20:4ω6, 20:4ω3, 20:5ω3, 22:1ω9, 22:5ω6, 22:5ω3, 22:6ω3.

The lipids used may be derived from animal or plant sources, or may beartificially synthesized.

Preferably, when the composition comprises a liquid diluent or thecomposition in tablet form is dissolved in a liquid diluent, thecomposition forms an emulsion or dispersion. Such compositions have anaqueous phase, which may contain one or more of the following; watersoluble vitamins, minerals, carotenoids, amino acids, peptides,nucleotides and polysaccharides. Any one of lipids, fat solublevitamins, carotenoids, minerals, peptides, nucleotides and amino acidsmay be contained in micelle or “microencapsulated” form, preferablydistributed evenly throughout the composition. The presence of themicelles has been shown to aid the uptake of fats and fat solublevitamins, carotenoids and amino acids from the diet at the level of thedigestive tract. This, combined with the simultaneous presentation ofwater soluble vitamins, minerals, peptides, nucleotides, polysaccharidescarotenoids and/or amino acids, has a synergistic effect on thebioavailability of the composition. Preferably, the emulsions ordispersions are formed by high speed blending.

The high level of bioavailability is partly due to the fact that thereis a reduced potential for chemical interactions in such preparations.Indeed, it has been noted that there is a reduction in the oxidation ofvitamins and carotenoids of these liquid compositions in the postapplication stage.

In a further preferred embodiment the composition comprises one or morewater soluble vitamins and one or more fat soluble vitamins.

In a further preferred embodiment the composition comprises one or morewater soluble carotenoids and one or more fat soluble carotenoids.

In a further preferred embodiment the composition comprises one or morewater soluble amino acids and one or more fat soluble amino acids.

In a further preferred embodiment the composition comprises one or morewater soluble minerals and one or more fat soluble minerals.

In a further preferred embodiment the composition comprises one or morewater soluble peptides and one or more fat soluble peptides.

In a further preferred embodiment the composition comprises one or morewater soluble nucleic acids and one or more fat soluble nucleic acids.

The polysaccharide is preferably a non-starch polysaccharide and mostpreferably a glucan. Preferably, 1,3 β-glucan, or 1, 6 β-glucan arecontemplated since it has been shown that these molecules have anon-specific immunomodulatory role, particularly in fish physiology.

Cellulose, gum and sugar derivatives may be added to the composition toaid dispersion within or onto feeds by virtue of their ability toincrease solution viscosity and adherence. These, however, are notessential and are not required for emulsification of this composition.Indeed, in the absence of such cellulose, gum or sugar derivatives, thecomposition is still capable of adhering surprisingly well to feed.Thus, a preferred composition of the invention does not contain gum,cellulose, sugar and/or dextrin.

Gelling agents, or combinations of gelling agents, may also be includedin the composition so as to form a gel preparation. Suitable gellingagents would be known in the art, such as locust bean gum, zanthan gum,natural binding agents derived from plants or algae, pectins, starch,cellulose derivatives such as carboxy-methyl-cellulose, gelatine, agar,or carrageenan.

The composition may additionally include one or more emulsifier, one ormore antioxidants other than a carotenoid, one or more preservatives,one or more stabilising agents and/or one or more particulate materials.

The emulsifying agents, such as Polysorbate 80, help in the formation ofthe micelle “microencapsulated” fat soluble components. Alternatively,or in addition, the micelles may be formed by high speed blending.

The inclusion of stabilising agents, such as monopropylene glycol, inthe composition help stabilise the fat soluble components and optimisemicelle distribution. The use of such stabilising agents reducespotential for product turbidity and affords excellent product clarity.

Preservatives, such as phosphoric acid or potassium sorbate, may beincluded in the composition to preserve the composition by preventingthe growth of bacteria, fungi and yeasts.

The addition of antioxidants to the composition aids stability. Examplesof suitable antioxidants include ascorbyl polyphosphate and butylatedhydroxy-toluene. Antioxidants prevent or minimize the loss of the activecomponents of the composition, thereby extending the shelf life of thecomposition and providing protection to the finished product in the postapplication phase.

The particulate material may take the form of an inert particulate orcan be formed from one or more of the carotenoids (e.g. from Phaffiarhodozymax or Haematoccocus algae), vitamins, minerals (such asselenium), beta glucans, or peptides of the composition. These particlesmay act as carriers for the other components of the composition and havebeen shown to be particularly effective at absorbing components of thecomposition that are prepared in micelle form.

Compositions of this sort are particularly preferred for enriching livefeed that are capable of ingesting the particulate matter (e.g. 12 hourpost hatching artemia or mysis and daphnia). Such feed are capable ofloading their gastrointestinal tract lumen with the composition where itis not immediately subjected to biochemical breakdown.

It is preferred that the substances for inclusion in the composition canbe ingested, without experiencing any toxic effects, by the species thatis intended to consume the composition.

In a further preferred embodiment of the invention, the inclusion of oneor more carotenoid in the composition is optional.

It should be realised that the amounts of carotenoid, vitamin, mineral,amino acid, lipid, peptide, nucleotide or polysaccharide as well asemulsifier, antioxidant, preservative and stabilising agent are dictatedby a number of functions, namely the form of preparation (dry, fluid,encapsulated), the particular species and age of the animal intended toingest the composition, and the deficiencies in their diet. Accordingly,the skilled person would be able to determine the appropriate amounts inthese specific circumstances.

Not wishing to be limited further, but in the interests of clarity, thefollowing are examples of suitable ranges for the amounts of componentspresent in the compounds of the invention.

Carotenoids may be present in between 0-99, 0-95, 0-85, 0-80, 50-95,80-95, 0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of thecomposition, not including any aqueous diluent.

Vitamins may be present in between 0-99, 0-95, 0-85, 0-80, 50-95, 80-95,0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of the composition,not including any aqueous diluent.

Minerals may be present in between 0-99, 0-95, 0-85, 0-80, 50-95, 80-95,0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of the composition,not including any aqueous diluent.

Amino acid may be present in between 0-99, 0-95, 0-85, 0-80, 50-95,80-95, 0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of thecomposition, not including any aqueous diluent.

Lipids may be present in between 0-99, 0-95, 0-85, 0-80, 50-95, 80-95,0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of the composition,not including any aqueous diluent.

Peptides may be present in between 0-99, 0-95, 0-85, 0-80, 50-95, 80-95,0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of the composition,not including any aqueous diluent.

Nucleotide may be present in between 0-99, 0-95, 0-85, 0-80, 50-95,80-95, 0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of thecomposition, not including any aqueous diluent.

Polysaccharide may be present in between 0-99, 0-95, 0-85, 0-80, 50-95,80-95, 0-25, 0-10, 0-5, 0.1-1, 0.001-1, or 0.0001-1% Wt/Wt of thecomposition, not including any aqueous diluent.

Emulsifier may be present in between 0-55, 0-65, 0-45, 0-35, 0-25, 0-10,0-5, 5-10, 5-20, 10-30, 20-40, 0.01-1, 0.001-1, or 0.0001-1% Wt/Wt ofthe composition, not including any aqueous diluent.

Antioxidant may be present in between 0-55, 0-65, 0-45, 0-35, 0-25,0-10, 0-5, 5-10, 5-20, 10-30, 20-40, 0.01-1, 0.001-1, or 0.0001-1% Wt/Wtof the composition, not including any aqueous diluent.

Preservative may be present in between 0-55, 0-65, 0-45, 0-35, 0-25,0-10, 0-5, 5-10, 5-20, 10-30, 20-40, 0.01-1, 0.001-1, or 0.0001-1% Wt/Wtof the composition, not including any aqueous diluent.

Stabilising agents may be present in between 00-99, 0-95, 0-85, 0-80,50-95, 80-95, 0-25, 0-10, 0-5, or 0.0001-1% Wt/Wt of the composition,not including any aqueous diluent.

As discussed above, some compositions of the invention do not include anaqueous diluent. The other compositions that do contain an aqueousdiluent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02,0.01, 0.001 or 0.0001 litres of diluent per 1 kg of the other componentsof the composition.

The compositions of the invention are particularly useful as they can beused to enrich a diet in a multitude of ways, allowing the method ofenrichment to be chosen so as to best accommodate the species, or basicfeed of choice.

Thus, in a preferred embodiment the composition is used for enrichingthe diet of a captive species. Preferably the captive species are fishand more preferably the captive species are farmed fish, ornamental fishor aquarium fish.

In a further preferred embodiment the composition is incorporated infeed, examples of the method of incorporation are given below.

The composition can be added to feeds during or post manufacture,harvesting, processing, or delivery to the consumer. Examples ofsuitable feeds are; fish, crustaceans, artemia, copepods, mysis, krill,polychetes such as ragworm and lugworm, and farmed insects such ascrickets, mealworms and locusts. These feeds are particularly useful asfeeds for fish and reptiles. The composition may also be fed to theanimal in isolation from other food.

Thus, in accordance with a second aspect of the present invention, acomposition in accordance with the first aspect is used in a method forenriching feed by soaking the feed in the composition. In a preferredembodiment the feed is defrosting or defrosted. Alternatively, the feedis soaked in the composition prior to freezing. This method has beenshown to provide surprising levels of absorption and retention of thecomposition in feed. Even after soaking in a liquid formulation of thecomposition for as little as 30 minutes, followed by salt water washing,feed such as krill, mysis and brineshrimp have been shown to retain thecomposition.

Generally longer periods of soaking provide better absorption andretention of the composition in the feed. Accordingly, by controllingthe length of time the feed is soaked for, the user may control theamount of composition retained in the feed.

Soaking artemia in the composition six hours after hatching has beenshown to significantly increase the lipid content of the artemia. Thisis important as the lipids are vital in the provision of nutritionalrequirements of juvenile animals. The person skilled in the art would beaware how to adapt the lipid profile of he invention to suit theindividual needs of the target animal.

In accordance with a third aspect of the present invention, acomposition in accordance with the first aspect of the invention is usedin a method for enriching feed by spraying the feed with thecomposition. The composition may be sprayed onto feed such as processedfeeds (for example, extruded pellets) or the exoskeleton ofinvertebrates (such as crickets, or locusts). Greater penetration of thefeed may be achieved by using a pressure spray.

In accordance with a fourth aspect of the present invention, acomposition in accordance with the first aspect of the invention is usedin a method for enriching feed by adding the composition before orduring production of processed feed. In this way the composition ismixed through the feed whilst the feed itself is being produced. Thismethod of enrichment is preferably carried out prior to extrusion andshaping and/or prior to freezing of the processed feed. Greaterpenetration of the feeds may be achieved in the second, third and fourthaspect of the present application by applying a vacuum to the enrichedfeed or carrying out the method in a pressure vessel.

In accordance with a fifth aspect of the present invention, acomposition in accordance with the first aspect of the invention is usedin a method for enriching feed by injection of the composition into thefeed. This method is particularly useful for enriching feed in the formof fish for sharks and rays.

In accordance with a sixth aspect of the present invention, acomposition in accordance with the first aspect of the invention is usedin method for enrichment of feed by adding the composition to theenvironment or diet of live feed. In this way the live feed will eitherbe coated in the composition, or absorb or ingest the composition,thereby enriching the gut and body tissue of the live feed. If the livefeed is an aquatic species the composition may be added to the water inwhich the live feed are contained.

The composition may also be added to the environment of the animalintended to benefit from the composition. For example, if the animal isan aquatic species the composition may be added to the water in whichthe animal is contained. Thus, the animal will either ingest or absorbthe composition.

In accordance with a seventh aspect of the present invention, a feedcomprising a composition in accordance with a first aspect of theinvention is contemplated.

It has been found that the compositions of the present inventionprovides a protective environment for the feed during and after theenrichment process. For example, mysis shrimp typically degrades in 2-3hours after defrosting. After enrichment with the composition, thetreated mysis shrimp may be stored for 8-12 hours. This obviouslyincreases the ease of feeding as a single batch of feed can bedefrosted, enriched and stored for feeding to the animals throughout theday. Without this composition feed would have to be preparedperiodically throughout the day.

Feed may be pre-treated with enzymes such as proteases and/or lipasesprior to the enrichment of the feed with the composition (this isparticularly effective for those methods that involve soaking orspraying of the feed). Such enzymes alter the surface structure of feedsto allow more efficient enrichment by the composition of the presentinvention. Alternatively the composition may comprise aforementionedenzymes and the aforementioned pre-treatment step may be dispensed with.

The pH of the composition may also be varied in order to optimise theenrichment of specific feed.

The composition may be prepared in a kit form which could optionallycomprise enzymes and/or feed. Alternatively the enzymes and/or feed mybe provided in discreet portions.

The kit may include a vacuum or pressure device in order to furtherassist the enrichment process. In a preferred embodiment the packagingof the kit includes such a device so that the enriched feed may beeasily prepared under pressure or in a vacuum within the packaging.

Specific compositions according to the present invention will now bedescribed, by way of example only.

EXAMPLE 1

Material % (Wt/Wt) Phosphoric Acid (85% Food Grade) 2.6386 BHT P/L0.0264 Monopropylene Glycol BP/000 (P/L) 79.1583 Polysorbate 80(Alkamuls T80) 13.1930 Potassium Sorbate Powder BP 2.6386 PotassiumIodide BP-USP (Nutec) 0.0007 Panthenol-D (P/L) 0.0066 Vitamin APropionate 2.5 MIU 0.0165 Vitamin D3 Oil 4 MIU-G (P/L) 0.0007 Vitamin K0.0063 Biotin USP Pure 0.0007 Choline Chloride 05 BP 0.0132 Inositol(P/L) 0.0007 Nicotinamide (Nutec - P/L) 0.0693 Para-Amino-Benzoic Acid(P/L) 0.0660 Pyridoxine Hydrochloride (Nutec - P/L) 0.0073 Vitamin B1(Thiamine HCL) (P/L) 0.1649 Vitamin B2 (Riboflavin 5) (P/L) 0.0660Vitamin B12 Crystalline (P/L) 0.0066 Vitamin E Oil 93% FG 0.4947 VitaminC (as Ascorbyl Polyphosphate (Stay C)) 1.3193 Bioastin Oleoresin (COS)0.1135 Lucantin Pink (COS) 0.0508

The final product is diluted in purified water as required. For example,when 4 kg of the phosphoric acid is used the final product is diluted inpurified water to a final volume of 400 litres. These values for thefinal product include overage to ensure adequate amounts of thecomponents over a 18 month period.

EXAMPLE 2

Material % (Wt/Wt) Phosphoric Acid (85% Food Grade) 2.6430 BHT P/L0.0264 Monopropylene Glycol BP/000 (P/L) 79.2885 Polysorbate 80(Alkamuls T80) 13.2148 Potassium Sorbate Powder BP 2.6430 Potassiumiodide BP-USP (Nutec) 0.0007 Panthenol-D (P/L) 0.0066 Vitamin APropionate 2.5 MIU 0.0165 Vitamin D3 Oil 4 MIU-G (P/L) 0.0007 Vitamin K0.0063 Biotin USP Pure 0.0007 Choline Chloride 05 BP 0.0132 Inositol(P/L) 0.0007 Nicotinamide (Nutec - P/L) 0.0694 Para-Amino-Benzoic Acid(P/L) 0.0660 Pyridoxine Hydrochloride (Nutec - P/L) 0.0073 Vitamin B1(Thiamine HCL) (P/L) 0.1652 Vitamin B2 (Riboflavin 5) (P/L) 0.0073Vitamin B12 Crystalline (P/L) 0.0066 Vitamin E Oil 93% FG 0.4956 VitaminC (as Ascorbyl Polyphosphate (Stay C)) 1.3215

The final product is diluted in purified water as required. For example,when 4 kg of the phosphoric acid is used the final product is diluted inpurified water to a final volume of 400 litres. These values for thefinal product include overage to ensure adequate amounts of thecomponents over a 18 month period.

EXAMPLE 3

Material Amount Vitamin B3 10000 mg/kg Vitamin B6 1000 mg/kg Vitamin B21000 mg/kg Vitamin B1 24000 mg/kg Vitamin B12 1280 mg/kg Vitamin A5300000 iu/Kg Vitamin D3 245500 iu/kg Vitamin E 86000 iu/kg Vitamin C(as ascorbyl polyphosphate (Stay C)) 180000 mg/kg Vitamin K 1022 mg/kgPantothanate 850 mg/kg Choline 1000 mg/kg Folic Acid 5460 mg/kg Inositol29 mg/kg Biotin 23 mg/kg Iodine 26.1 mg/kg

Fe Gluconate may be added to the formulation at the rate of 17400 mg/kgas a source of dietary iron.

Marine algae may be added to the specification. These will supply arange of natural minerals and trace elements in addition to naturalsources of proteins, lipids and carbohydrates. These include Glucides,mannitol, alginates and cellulose. Natural aglae are also a source ofvitamins and may be used to supply some of the vitamins in theformulation.

Minerals supplied may include:

-   -   Calcium    -   Magnesium    -   Potassium    -   Sodium    -   Phosphorus    -   Sulphur    -   Iodine    -   Zinc    -   Manganese    -   Iron    -   Copper    -   Molybdenum    -   Selenium    -   Boron    -   Chromium    -   Nickel    -   Tin    -   Vanadium    -   Silica

Manufactured minerals and trace elements may be added to the formulation

EXAMPLE 4

Includes the components provided in Example 1, including BioastinOleoresin (COS) and Lucantin Pink (COS), but does not include the listedvitamins and minerals.

EXAMPLE 5

A composition as disclosed in Example 4 but 5 times more concentrated.

A study has been carried out in order to evaluate the ability of thecompositions of the present invention (specifically, examples 1, 4 and5) to enhance poor levels of carotenoid in frozen marine diets (TMCBrineshrimp, Mysis and Krill).

The compositions provided in examples 1, 4 and 5 were tested as follows:Test 1: 30 g of frozen marine feed were placed in a large weighing boat,to this 30 ml of the composition was added. After 30 minutes, 10 g offeed material was removed from the solution and blotted with absorbentmaterial to remove any surface composition. 1 g of the blotted feedmaterial in triplicate was then placed into test tubes.

A further 3 g of the blotted feed material was placed in a tea strainerand immersed in Tropic Marine seawater (1.024 @ 24° C.) for five secondswithin a slightly turbulent flow. The contents were then blotted again,and a further 1 g in triplicate of test material was placed in testtubes.

The same procedure as above was repeated except the wash phase was for15 seconds.

Using standard methods, each sample was analysed for total astaxanthin(a pre-hydrolysis of astaxanthin esters was used to base all findings ona ‘free’ astaxanthin basis). All samples were then run on a HPLC todetermine astaxanthin content by an established method. Moisture contentof the frozen marine diets was also established using the A.O.A.C (1990)methodology.

FIG. 1 (Brineshrimp subjected to soaking in the composition andsubsequent immersion in seawater.) The first frozen marine diet testedwas brineshrimp; this is the most widely used frozen feed supplement fortropical marine species. The brineshrimp tested had no trace ofastaxanthin although there may have been traces of β-carotene (notconfirmed by using beta carotene standard, but based on retention timesof this particular carotenoid would suggest this was the carotenoidpresent). FIG. 1 clearly demonstrates the potential of all thecompositions tested, each considerably boosting astaxanthin levels inthe brineshrimp. TABLE 1 (data presented in FIG. 1) Example 5 Example 4Example 1 Brineshrimp (test procedure) Ax present in μg/g Ax present inμg/g Ax present in μg/g Untreated brineshrimp n/f n/f n/f 30 minutessoak time in product 2.14 ± 0.71 0.33 ± 0.07 0.46 ± 0.05 30 minutes + 5second wash 0.89 ± 0.11 0.14 ± 0.06 0.21 ± 0.03 30 minutes + 15 secondwash 0.63 ± 0.23 0.16 ± 0.03 0.18 ± 0.04 3 hour soak time in product2.70 ± 0.18 0.40 ± 0.01 0.46 ± 0.07 3 hour + 5 second wash 2.01 ± 0.450.38 ± 0.04 0.50 ± 0.08 3 hour + 15 second wash 2.03 ± 0.05 0.38 ± 0.030.39 ± 0.07

FIG. 2 (Krill subjected to soaking in the composition and subsequentimmersion in seawater.) The second test was completed on frozen Krill.The main pigment found in krill is astaxanthin, although othercarotenoid pigments are also found. The level of astaxanthin can varyamong different krill products, but generally it is between 150-200 ppmon a dry weight basis. Astaxanthin is present generally in theesterified form. In contrast, synthetic astaxanthin, which is widelyused in aquafeeds, is exclusively found in a non-esterified form. It isthought that the esterified form of astaxanthin must be converted to thefree form prior to being absorbed from the gut. TABLE 2 (data presentedin FIG. 2) Example 5 Example 4 Example 1 Krill (test procedure) Axpresent in μg/g Ax present in μg/g Ax present in μg/g Untreated krill 2.96 ± 0.54  2.96 ± 0.54  2.96 ± 0.54 30 minutes soak time in product23.25 ± 0.34  9.92 ± 3.06 12.95 ± 4.44 30 minutes + 5 second wash 15.97± 4.20 13.45 ± 0.84 12.38 ± 2.77 30 minutes + 15 second wash 19.37 ±0.75 11.30 ± 3.35 11.33 ± 0.70 3 hour soak time in product 40.40 ± 2.8512.45 ± 2.10 16.09 ± 3.94 3 hour + 5 second wash 43.08 ± 0.60 13.69 ±3.08 18.53 ± 1.61 3 hour + 15 second wash 47.72 ± 3.20 14.06 ± 0.8616.25 ± 0.38

The second biggest astaxanthin enhancement was achieved using frozenkrill in conjunction with the tested compositions. Astaxanthin levelswere elevated to almost eleven times the concentration in the basalfrozen diet. This is depended on the composition used and the soak time.

FIG. 3 (Krill subjected to soaking in the composition and subsequentimmersion in seawater.)The final test focused on mysis shrimp. Similarto the brineshrimp they have a very poor carotenoid profile (analysis byHPLC confirmed this). The results detailed in FIG. 3 and table 3 showexcellent enhancement with the tested compositions even after washing ofthe material in general. TABLE 3 (data presented in FIG. 3) Example 5Example 4 Example 1 Mysis (test procedure) Ax present in μg/g Ax presentin μg/g Ax present in μg/g Untreated mysis n/f n/f n/f 30 minutes soaktime in product 15.90 ± 1.77 1.92 ± 0.24 3.16 ± 0.25 30 minutes + 5second wash  7.79 ± 0.53 1.24 ± 0.04 2.49 ± 0.22 30 minutes + 15 secondwash  6.60 ± 0.66 1.31 ± 0.19 2.75 ± 0.23 3 hour soak time in product20.22 ± 1.74 1.74 ± 0.37 3.46 ± 0.63 3 hour + 5 second wash 14.35 ± 0.631.64 ± 0.08 3.12 ± 0.42 3 hour + 15 second wash 13.14 ± 1.16 1.40 ± 0.333.15 ± 0.21

The results of these tests clearly demonstrate that there is retentionof astaxanthin (carotenoid) in the tissue matrix of various marinezooplankton and invertebrate organisms. Accordingly the compositions ofthe current invention are particularly effective enrichment products fornatural feed for marine/fresh water fish species.

Astaxanthin in the compositions are mainly in the esterified form(derived from for example Haematoccocus pluvalis) and is more effectivethan the synthetic ‘free’ form. Astaxanthin esterified to fatty acidacyl groups confer superior adsorption properties for tissues containinglipids as found in krill, mysis and adult brineshrimp.

The highest level of absorption and retention was attained in de-thawedfrozen krill and the least adsorption was found in brineshrimp.

It should be stated that a significant background level of astaxanthinwas measured in krill before soak treatment of the tested compositions.This was taken into consideration and is displayed in FIG. 2. Mysis hadno prior astaxanthin level but responded well to the soaking treatmentresulting in very good retention of carotenoid.

1-39. (canceled)
 40. A composition comprising one or more carotenoids,and one or more of the following substances: vitamins, minerals, aminoacids, lipids, peptides, nucleotides and/or polysaccharides.
 41. Acomposition as claimed in claim 40, further comprising an aqueousdiluent, wherein the composition is in the liquid state.
 42. Acomposition as claimed in claim 40, wherein one or more of thecomponents of the composition are water soluble.
 43. A composition asclaimed in claim 40, wherein one or more of the components of thecomposition is/are fat soluble.
 44. A composition as claimed claim 41,wherein the fat soluble components are in the form of micelles.
 45. Acomposition as claimed in claim 40, wherein the composition comprisesone or more water soluble vitamins and one or more fat soluble vitamins.46. A composition as claimed in claim 40, wherein the compositioncomprises one or more water soluble carotinoids and one or more fatsoluble carotinoids.
 47. A composition as claimed in claim 40, whereinthe composition comprises one or more water soluble amino acids and oneor more fat soluble amino acids.
 48. A composition as claimed in claim40, wherein the composition comprises one or more water soluble mineralsand one or more fat soluble minerals.
 49. A composition as claimed inclaim 40, wherein the composition comprises one or more water solublepeptides and one or more fat soluble peptides.
 50. A composition asclaimed in claim 40, wherein the composition comprises one or more watersoluble nucleic acids and one or more fat soluble nucleic acids.
 51. Acomposition as claimed in claim 40, wherein the composition comprises anoil.
 52. A composition as claimed in claim 40, wherein the compositioncomprises water soluble 1, 3 β-glucan or 1,6 β-glucan.
 53. A compositionas claimed in claim 40, further comprising cellulose, gum and or a sugarderivative.
 54. A composition as claimed in claim 40, further comprisingan emulsifier, preferably Polysorbate
 80. 55. A composition as claimedin claim 40, further comprising a stabilising agent, preferablymonopropylene glycol.
 56. A composition as claimed in claim 40, furthercomprising a preservative, preferably phosphoric acid and/or potassiumsorbate.
 57. A composition as claimed in claim 40, further comprising anantioxidant other than a carotenoid, preferably ascorbyl polyphosphateand/or butylated hydroxy-toluene.
 58. A composition as claimed in claim40, wherein the inclusion of one or more carotenoid is optional.
 59. Acomposition for enriching the diet of a captive species, wherein thecomposition is as claimed in claim
 40. 60. A composition as claimed inclaim 59, wherein the captive species are fish, preferably farmed fish,ornamental fish, or aquarium fish.
 61. A composition as claimed in claim59, wherein the composition is incorporated in feed.
 62. A method forthe enrichment of feed, comprising soaking the feed in a composition asclaimed in claim
 40. 63. A method as claimed in claim 62, wherein thefeed is defrosting or defrosted.
 64. A method as claimed in claim 62,wherein the feed is soaked in the composition prior to freezing.
 65. Amethod for the enrichment of feed, comprising spraying the feed with acomposition as claimed in claim
 40. 66. A method for the enrichment offeed, comprising the addition of the composition as claimed in claim 40before or during the production of processed feed.
 67. A method forenrichment of feed, comprising the injection of the composition asclaimed in claim 40 into the feed.
 68. A method for enrichment of feed,comprising adding the composition as claimed in claim 40 to theenvironment or diet of live feed.
 69. A feed comprising a composition asclaimed in claim 40.